Abstract 18126: Ectodomain Shedding and Autocleavage of the Cardiac Membrane Protease Corin
Corin is a membrane serine protease expressed primarily in the heart, where it regulates blood pressure and cardiac function by activating natriuretic peptides. Recently, soluble corin was detected in human plasma and its levels were significantly lower in patients with heart failure (HF), suggesting that corin is cleaved from the cell surface and that plasma soluble corin may be used as a diagnostic biomarker for HF. In this study, we examined the molecular mechanism responsible for corin ectodomain shedding. In both transfected HEK 293 cells and HL-1 cardiomyocytes, we detected a 180-kDa corin fragment in the conditioned medium by immunoprecipitation and Western analysis. When the cells were incubated with ADAM protease inhibitors, this corin fragment was undetectable, indicating that the shedding of this corin fragment was mediated by an ADAM protease(s). When the cells were treated with phorbol myristate acetate or ionomycin, the corin shedding was increased by ∼2 fold, suggesting that ADAM17 is a likely corin sheddase. By RT-PCR, we confirmed ADAM17 mRNA expression in both cell lines. By analyzing the shedding of series of point or deletion mutants, we showed that the ADAM cleavage site was located in a 29-amino acid segment in the corin juxtamembrane domain. In functional studies, the 180-kDa soluble corin was active, converting pro-atrial natriuretic peptide (pro-ANP) to ANP. In addition to the ectodomain shedding, we also identified two corin autocleavage sites: one at R164 in Frizzled domain 1 and the other at residue R427 in LDLR5 repeat. In the presence of serine protease inhibitors or by site-directed mutagenesis at the corin active site S985, corin autocleavage was prevented. The autocleaved corin fragments had little pro-ANP processing activity in functional assays, suggesting that corin autocleavage may act to limit its activity. Together, our results show that corin is cleaved from the cell surface by either ADAM-mediated shedding or autocleavage. These proteolytic events may represent different mechanisms regulating corin cell surface expression and activity. Altered corin ectodomain shedding and/or autocleavage may play an important role in HF.
- © 2010 by American Heart Association, Inc.